Mycoplasmas are wall-less eubacteria which exist as parasites within eucaryotic cells. They are the smallest, simplest, and most primitive form of self-replicating procaryotes, consisting of only three major components--a plasma membrane, ribosomes, and a double-stranded genome of 75-150 kB. The limited biosynthetic capacity of these organisms accounts for their dependence on host cells as a source of nutrients. Mycoplasma are phylogenetically related to gram-positive bacteria such as Bacillus spp. and Clostridium spp. Their size, 300 to 800 nm in diameter, is similar to that of a large virus. Mycoplasmas can be either coccoid, helical, or filamentous in shape.
Mycoplasma are generally characterized on the basis of their host association. In man, infection with respiratory mycoplasmas can lead to a benign, self-limiting, moderately troublesome disease which may yield pulmonary, gastrointestinal, cardiac, and hematological complications. A different set of mycoplasmas are responsible for infections of the human urogenital tract, and an attendant set of complications. In contrast to those mycoplasmas which infect human cells, there is a distinct group of mycoplasma strains which are associated with the infection of cell lines grown in tissue culture. While these infections pose no health threat to the researcher working with the infected cell, they can have major impact on the normal physiology and structural integrity of the infected cell. Table 1, below, describes the range of effects which mycoplasma can have on cell cultures. Because of the breadth of cellular responses which are influenced by mycoplasma infections, researchers must question the validity of any experimental results obtained with infected cells. This concern over the effects of mycoplasma infections on cell responses has provided the impetus for developing methods for detection of infection
TABLE 1 ______________________________________ Effects of Mycoplasmas on Cell Cultures ______________________________________ Interference with the growth rate of cells Inhibition of lymphocyte transformation Stimulation of lymphocyte transformation Induction of morphological alterations, including cytopathology Altered DNA, RNA, and protein synthesis Alterations of ribosomal RNA profiles Alteration of enzyme patterns Interference with selection of mutant mammalian cells Induction of chromosomal aberrations Depletion of the essential amino acid arginine from cell culture growth medium Inhibition of virus yields Enhancement of virus yields Co-purification of mycoplasmas with cell organelles (e.g., mitochondria) Redistribution and modification of host cell plasma membrane antigens Apparent reduction in tumorigenic potential of malignant ______________________________________ cells
in cell cultures. Numerous techniques for the detection of mycoplasma have been described. They have been based on microbiological, biochemical, biophysical, and microscopic detection systems. The strengths and weaknesses of each of these techniques has been comprehensively reviewed, "Gene Probe Detection of Human and Cell Culture Mycoplasmas," by Dular, in Gene Probes for Bacteria, Academic Press, San Diego (1990). There are two detection techniques which have been found to be the most reliable indicators of mycoplasma infection. The first of these is the MycoTect.TM. kit marketed by Gibco. This kit is based on the fact that mycoplasma express the gene for adenosine phosphorylase, and as such, will metabolize the suicide substrate 6-methylpurine deoxyriboside into 6-methylpurine and 6-methylriboside, two compounds which are toxic for eucaryotic cells. In this test, cell-free culture supernatants taken from test cells are mixed with the suicide substrate, and then added to cultures of the non-infected indicator cell line 3T6. After several days, the indicator cells are inspected for viability, and cell death is used as a indicator of mycoplasma infection in the test cells.
The other test for mycoplasma which has gained wide acceptance is the rRNA hybridization probe technology of GenProbe.TM.. This test uses a radiolabeled complementary DNA "specific" for mycoplasma to probe for the presence of mycoplasma rRNA. Cell-free culture supernatants taken from test cells are incubated with the radiolabeled probe. The free probe is separated from the bound probe using a hydroxyapatite column; the duplexed probe comes out in the column void volume. The detection of radioactivity in the void above a certain threshold value is indicative of mycoplasma infection. New versions of the GenProbe.TM. kit (Mycoplasm T.C. II) use a centrifugation and washing step, instead of a column, to separate free radioactivity from bound radioactivity.
Each of the currently accepted tests have drawbacks to their technology. The MycoTect.TM. detection system requires that the investigator culture his/her test cells in the absence of antibiotics for several passages before culture supernatants can be taken for testing. Because this test is based on the response of an indicator cell line, it is several days (4-5 days) before the results are obtained. Both versions of the GenProbe.TM. probe test require a separation step which is time-consuming and because the readout utilizes radioactivity, it generates radioactive waste.
Because of the shortcomings of these tests, it is of interest to develop an improved test system for the detection of mycoplasma that avoids these shortcomings. Accordingly, a new sensitive, convenient and efficient test system for the detection and identification of mycoplasma is described herein.